Protective Effects Of Cytochrome C On Renal Tubular Toxicity Induced By Gentamicin And Etimicin In Rats

Aminoglycosides (AGs) such as gentamicin and etimicin are widely used in thetreatment of severe and complicated bacterial infections, especially those caused byGram-negative pathogens. Unfortunately, they can easily cause nephrotoxicity whichseriously restricts their optimal use in the clinical applications. Increasing evidence hasrevealed that the mechanism of AGs-induced nephrotoxicity may be apoptosis ofproximal tubular epithelial cells induced by their accumulation in kidneys.Cytochrome C, a water-soluble chromoprotein that carries an iron porphyrin group, isprimarily extracted from pig hearts that clinically serves as a cellular respiratorystimulant and offers valuable assistance in emergent or adjunctive treatment of a varietyof tissue hypoxia, such as carbon monoxide poisoning, hypnotic intoxication, severehypoxia in shock stage, dyspnea, myocardial anoxia, etc. Reportedly, Cytochrome C at adose of80mg/kg can decrease the amount of low-dose gentamicin of0.1mg/kgaccumulated in rat kidneys; at100mg/kg can significantly inhibit urinary excretion ofN-acetyl-beta-D-glucosaminidase (NAG), an early biomarker of renal tubular injury, ingentamicin30mg/kg-treated rats.Only one published study so far has demonstrated that Cytochrome C can effectivelyreduce gentamicin accumulation in renal tissues, but the dose of gentamicinadministered in the study was too low for clinical practice. To provide usefulinformation for clinical application, in the present study we investigated the possibleprotective effects of Cytochrome C on nephrotoxicity induced by usual and/or largedoses of gentamicin or etimicin in vivo from the angle of apoptotic injury and histopathological changes in tubular epithelial cells.This study consists of the following two sections.1. Protective effect of Cytochrome C against gentamicin-induced renal tubulartoxicity in ratsHealthy male Wistar rats were randomly divided into vehicle control group,gentamicin group and gentamicin plus Cytochrome C group, with10animals in each.Rats in the former2groups were given i.p. normal saline solution and gentamicin100mg/kg/d respectively, in the third group received caudal vein injection of Cytochrome C100mg/kg/d30min before gentamicin administration (100mg/kg/d, i.p.). Each rat wassacrificed24hours after the single dosing and both kidneys were harvested, the contentof gentamicin in the kidney was determined by fluorescence polarization immunoassay(FPIA) technique in the latter two groups. The apoptotic renal tubular epithelial cellswere detected by in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTPnick end labeling (TUNEL) assay, and the positive numbers of apoptotic cells in eachgroup were calculated by Color Medical Image Analysis System-II (CMIAS-II). Thehistopathological changes of renal tissues were observed by HE staining. Results of thestudy showed that, compared with the vehicle control group, the positive number ofapoptotic tubular epithelial cells in the gentamicin group increased significantly (P＜0.01); compared with the gentamicin group, the content of gentamicin in rat kidneys ofthe gentamicin plus Cytochrome C group declined by33.86%(P＜0.05), the positivenumber of apoptotic cells decreased by36.53%(P＜0.05); renal tubular epithelial cellsswelling, vacuolar degeneration, tubulointerstitial inflammatory cell infiltration andglomerulus engorgement and swelling were all improved to some extent. The studyindicated that Cytochrome C can protect against gentamicin-induced renal tubulartoxicity by reducing the amount of gentamicin accumulated in kidneys.2. Protective effect of Cytochrome C against etimicin-induced renal tubulartoxicity in rats Healthy male Wistar rats were assigned randomly into group1to8, including onevehicle control group (group1), one Cytochrome C control group (group2), threeetimicin groups (group3to5) and three etimicin plus Cytochrome C groups (group6to8) with10animals in each of the study groups. Rats in the group1received i.p. normalsaline solution alone, in the group2received Cytochrome C100mg/kg/d by the tailvein. Etimicin-treated animals were i.p. administered with etimicin10mg/kg/d (group3),30mg/kg/d (group4) or100mg/kg/d (group5) alone or in combination withCytochrome C100mg/kg/d via the tail vein30min before etimicin administration(group6to8, respectively). All animals were given the drugs for three consecutive days.24hours after the last injection, each rat was sacrificed and both kidneys were rapidlyremoved from the opened abdominal cavity. Etimicin accumulation in the kidneys wasmeasured by high performance liquid chromatography (HPLC) with pre-columnderivatization, renal tubular epithelial cell apoptosis was detected by TUNEL techniqueand the positive numbers of apoptotic cells were calculated by CMIAS-II software. Thepathological changes of renal tubule were examined by HE staining. The results showedthat compared with the vehicle control group or the Cytochrome C control group, thepositive numbers of apoptotic tubular epithelial cells in three groups treated withetimicin alone significantly increased (P＜0.01); compared with etimicin10,30and100mg/kg alone groups, pre-treatment with Cytochrome C100mg/kg decreased theamount of etimicin accumulated in rat kidneys by43.72%(P＜0.05),45.22%(P＜0.05)and12.81%(P＞0.05) respectively, the positive number of apoptotic tubular epithelialcells declined by63.57%(P＜0.01),68.55%(P＜0.01) and10.65%(P＞0.05)respectively. The histopathological changes of renal tissues, such as variable degrees ofglomerular atrophy and vascular congestion, tubular dilation, epithelial degeneration,vacuolization, cell atrophy and desquamation, mononuclear cell infiltration in renalcortex were improved to some extent when Cytochrome C was sequentiallyadministered. This study suggested that Cytochrome C can provide marked protective effect against etimicin-induced nephrotoxicity, apoptosis and pathological changes ofrenal tubular epithelial cells, by its inhibition of etimicin accumulation in kidneys.